The present invention relates generally to recombinant DNA technology; and more specifically, to methods of producing recombinant virions. In particular, by suppressing translation of a transgene during production of recombinant virions, the methods expand the number of transgenes that can be produced as recombinant virions.
Viral vectors capable of transferring genetic material into mammalian cells have the potential to provide a wide range of experimental and therapeutic uses. See, e.g., Jolly, Cancer Gene Therapy 1(1):51-64, 1994); U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. Viral vectors can be derived from a variety of viruses, including adenoviruses, retroviruses, and alphaviruses such as Sindbis (see, e.g., Strauss and Strauss, 1994, Microbio. Rev., 58:491-562). For a description of viral vectors and their uses, see, for example, Gene Therapy: Principles and Applications (T. Blankenstein, et., 1999, Springer-Verlag, Inc.) and Understanding Gene Therapy (N. Lemoine, ed., 2000, R-G Vile). 
In each of these viral vector systems, it is desirable to have high levels of transgene expression in target cells. However, transgene expression during virion production is not necessary, and, in many cases, interferes with viral growth. To circumvent this problem, several groups have attempted to regulate transcription of the transgene during production of adenoviral virions, for instance by operably linking the transgene to an inducible promoter. The promoter is kept inactivated during virion production and induced in the target cell. However, this transcriptional regulation imposes many constraints on experiments and therapies.
Thus, there remains a need for methods of suppressing transgene expression during virion production while maintaining high transgene expression in the target cell(s).
The present invention solves this and other problems by providing systems wherein expression of the heterologous transgene may be reduced (suppressed) in desired cells, including during the virion packaging process. The methods allow for the suppression of transgene translation in virion producing cells while maintaining the capacity for high level expression and translation of the transgene in all other cell types. The system described is applicable to a variety of viral vectors and, in addition, can be combined with other virion production systems, including, for example, adenovirus virion construction in a cre-lox system.
In one aspect the invention includes a host cell (e.g., a 293 cell) comprising a TOP-binding ligand and packaging elements. In certain embodiments, the TOP-binding ligand and/or one or more packaging elements is encoded by one or more polynucleotides while in other embodiments, the TOP-binding ligand and/or one or more of the packaging elements are introduced into the host cell via other means, such as injection or the like. Thus, introduction of polynucleotides encoding the desired gene product can be used in combination with other means of introducing molecules into the host cell. In certain embodiments, the host cell is stably or transiently transfected with a nucleic acid molecule encoding the TOP-binding ligand (e.g., an MS2 or R17 coat protein) and stably or transiently transfected with the polynucleotides encoding the packaging elements (e.g, E1A and E1B). In yet other embodiments, the host cell further comprises a cre recombinase.
In another aspect, the invention includes a method of producing recombinant virions comprising culturing a recombinant viral vector comprising a translational operator sequence (TOP) operably linked to a transgene with (i) a TOP-binding ligand and (ii) packaging elements; under conditions such that expression of the transgene is suppressed during production of the virions. In certain embodiments, the recombinant viral vector is an adenovirus vector, a retroviral (e.g., lentiviral vector such as FIV, HIV, HIV-1, HIV-2 and SIV), an alphaviral vector or an adeno-associated vector. The TOP-binding ligand can be, for example, an MS2 coat protein or an R17 coat protein. The TOP-binding ligand and/or the packaging elements may be encoded by a polynucleotide that is stably or transiently transfected into a host cell, for example a 293 cell that expresses an MS2 coat protein, an E1A protein and an E1B protein. In certain embodiments, any of the recombinant viral vectors described herein further comprise an mRNA 5xe2x80x2,5xe2x80x2,7-methyl guanosine triphosphate cap sequence (m7G-cap) and the TOP is located within about 60 base pairs or within about 30 base pairs of the mRNA m7G-cap sequence. In embodiments where the recombinant viral vector is derived from adenovirus, the recombinant adenoviral vector and the packaging elements can be cultured the presence of an E1, E3 deleted adenovirus construct having loxP sites flanking the packaging site and a cre recombinase.
In yet another aspect, the invention includes a viral vector (e.g., derived from an adenovirus, an alphavirus, a retrovirus such as a lentivirus, or an adeno-associated virus) comprising a transgene operably linked to a TOP sequence. In certain embodiments, the TOP sequence is derived from an MS2 or R17 bacteriophage.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.